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. 2022 Jun 10;3(6):371-387.e9.
doi: 10.1016/j.medj.2022.04.001. Epub 2022 Apr 13.

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection

Aravind Natarajan  1   2 , Soumaya Zlitni  1   2 , Erin F Brooks  2 , Summer E Vance  2 , Alex Dahlen  3 , Haley Hedlin  3 , Ryan M Park  1   2 , Alvin Han  4 , Danica T Schmidtke  4 , Renu Verma  5 , Karen B Jacobson  5 , Julie Parsonnet  6   7 , Hector F Bonilla  6 , Upinder Singh  5 , Benjamin A Pinsky  5   8 , Jason R Andrews  5 , Prasanna Jagannathan  4   6 , Ami S Bhatt  1   2
Affiliations

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection

Aravind Natarajan et al. Med. .

Abstract

Background: COVID-19 manifests with respiratory, systemic, and gastrointestinal (GI) symptoms.1, SARS-CoV-2 RNA is detected in respiratory and fecal samples, and recent reports demonstrate viral replication in both the lung and intestinal tissue.2, 3, 4 Although much is known about early fecal RNA shedding, little is known about long-term shedding, especially in those with mild COVID-19. Furthermore, most reports of fecal RNA shedding do not correlate these findings with GI symptoms.5.

Methods: We analyzed the dynamics of fecal RNA shedding up to 10 months after COVID-19 diagnosis in 113 individuals with mild to moderate disease. We also correlated shedding with disease symptoms.

Findings: Fecal SARS-CoV-2 RNA is detected in 49.2% [95% confidence interval, 38.2%-60.3%] of participants within the first week after diagnosis. Whereas there was no ongoing oropharyngeal SARS-CoV-2 RNA shedding in subjects at 4 months, 12.7% [8.5%-18.4%] of participants continued to shed SARS-CoV-2 RNA in the feces at 4 months after diagnosis and 3.8% [2.0%-7.3%] shed at 7 months. Finally, we found that GI symptoms (abdominal pain, nausea, vomiting) are associated with fecal shedding of SARS-CoV-2 RNA.

Conclusions: The extended presence of viral RNA in feces, but not in respiratory samples, along with the association of fecal viral RNA shedding with GI symptoms suggest that SARS-CoV-2 infects the GI tract and that this infection can be prolonged in a subset of individuals with COVID-19.

Funding: This research was supported by a Stanford ChemH-IMA grant; fellowships from the AACR and NSF; and NIH R01-AI148623, R01-AI143757, and UL1TR003142.

Keywords: COVID-19; SARS-CoV-2; fecal RNA; gastrointestinal infection; viral shedding.

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Conflict of interest statement

The authors declare no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
Summary of study protocol and cohort demographics (A) Sample and data collection timeline represented in days. Day 0 marks the day of enrollment in the trial, within 72 h of a COVID-19 diagnosis. Each sample collection event is marked by a colored dot, where orange represents a blood draw and blue an oropharyngeal (OP) swab. Additionally, clinical appointments and symptom surveys are marked by yellow and green dots, respectively. Some of these events are marked by day ranges to represent collection time frames. The symptom survey at day 0 retrospectively collected symptomatology for 3 weeks prior to enrollment using a single questionnaire. Symptom surveys at time points centered around days 120, 210, and 300 retrospectively collected symptomatology for 1 week prior to the appointment using a single questionnaire at each timepoint. Collection of stool samples and their respective day ranges are marked below the timeline. Subjects were asked to provide samples in the OMNIgene GUT collection tube (OG) and the Zymo DNA/RNA shield fecal collection tube (ZY) at six time points. (B) Cohort characteristics. 120 participants were enrolled in the clinical trial. Participants had a COVID-19 infection of mild to moderate severity and were between the ages of 18 and 71. The age and sex distributions of the paticipants are reprented here. The x axis separates the groups by self-reported sex, and the y axis lists age in years. Each bar represents a range of 5 years.
Figure 2
Figure 2
Fecal and oropharyngeal viral gRNA measurements over time (A) Summary of viral RNA positivity rates as determined by fecal and OP samples acquired from participants enrolled in the study for a period of around 28 days. The x axis lists time point categories since enrollment as days 3 (range 0–7), 14 (8–21), and 28 (22–35). The y axis lists the percentage of fecal samples (brown bar) and OP samples (gray bar) that tested positive at each of the time points. Fecal positivity rates are evaluated using the logistic GEE model described in the statistical methods section (see STAR Methods), which averages over all of the sample collection methods, gene types, and technical replicates. OP positivity rates are evaluated for the swab taken within 3 days of the fecal sample. Each bar also marks the 95% confidence interval (CI). Number of participants and percent positivity are listed as numbers at the top of the plot in black and red fonts, respectively, and summarized in Data S1. (B) Same as (A), except restricted to the subset of those who participated in the extended study, and following them through all six time points. As before, the x axis lists time point categories since enrollment: days 3 (range 0–7), 14 (8–21), 28 (22–35), 120 (75–165), 210 (166–255), and 300 (>255 days), and the y axis lists the percentage of participants with positive fecal samples (brown bar) and OP samples (gray bar) at each of the time points, with 95% CI. Number of participants and percent positivity are listed in black and red fonts and summarized in Data S1. (C) SARS-CoV-2 viral RNA concentration in stool samples collected in the ZY kit from participants (n = 104) with mild to moderate COVID-19 infection over a time period of 300 days from enrollment in the study. Note that the ZY kits had higher overall positivity rates than the OG kits, so positivity rates in this panel tend to be slightly larger than the numbers in the previous two panels, which average over kits and genes. Fecal viral RNA concentration was determined using RT-qPCR with primers/probes targeting the E, N1, N2, and RdRP genes in the SARS-CoV-2 genome, as indicated in the tab at the top of each panel. The x axis lists time point categories since enrollment. The y axis lists the percentage of participants with a given viral RNA concentration, as indicated by the color scheme in the stacked bar plot; dark blue refers to those with no detectable viral RNA, orange to viral RNA concentrations between 0 and one log10 copy/μL, yellow between one and two log10 copies/μL, green between two and three log10 copies/μL, and light blue over three log10 copies/μL. Number of participants per time point is listed above each bar in the stacked bar plot. (D) Fecal viral RNA concentration in stool samples collected in the ZY kit from participants (n = 104) with mild to moderate COVID-19 infection and assayed using RT-qPCR detecting the N1 gene (viral RNA concentration in log10 copies per μL) versus time (continuous variable; x axis). Time point categories are indicated by color scheme: yellow for days 3 (range 0–7), lavender for day 14 (8–21), red for day 28 (22–35), gray for day 120 (75–165), light blue for day 210 (166–255), and dark blue for day 300 (>255 days). A smoothed line generated using LOESS regression (span parameter = 0.75) and 95% CI is marked in the scatterplot. Note that all viral RNA concentration measurements are expressed on a logarithmic scale by applying the transformation log10 (viral RNA concentration+1).
Figure 3
Figure 3
The effect of IFN-λ on fecal viral RNA shedding (A) Percentage of participants with detectable fecal SARS-CoV-2 RNA across each of the study arms, as evaluated using the logistic GEE model described in the statistical methods section (STAR Methods). The x axis marks the time point in the study: days 3 (range 0–7), 14 (8–21), and 28 (22–35). The y axis indicates the percentage of participants with detectable fecal SARS-CoV-2 RNA. The blue bar corresponds to participants in the placebo control arm, and the orange bar corresponds to participants in the IFN-λ intervention arm. Each bar also marks the 95% CI. Number of participants and percentage of participants that provided a positive stool sample are listed above each stacked bar in black and red fonts, respectively, and summarized in Data S1. (B) Odds ratio comparing detectable fecal SARS-CoV-2 RNA shedding in the IFN-λ intervention arm with the placebo arm at each time point in the first month of the study. The x axis marks the odds ratio adjusted for age, sex, collection kit type (OG or ZY), and target gene (E, N1, N2, or RdRP) (aOR). The y axis marks the time point in the study: days 3 (range 0–7), 14 (8–21), and 28 (22–35). The point marks the aOR, flanked by lines denoting the 95% CIs. The red dashed vertical line at aOR = 1.0 indicates no association.
Figure 4
Figure 4
Association between fecal viral RNA shedding and symptoms We present these results in the overall population, as well as stratified by the presence and absence of ongoing viral RNA shedding from the oropharynx (OP). (A) Summary of the association between viral RNA shedding and report of a given symptom in all participants. Shedding and symptom data from up to day 28 were included in this analysis. Adjusted odds ratios (aOR) for this association were evaluated using the logistic GEE model described in the statistical methods section (STAR Methods), which averages over collection kits (OG and ZY), target genes (E, N1, N2, and RdRP) and technical replicates and is adjusted for age, sex, collection kit, and target gene. The x axis indicates the aOR for the presence of a given symptom. The y axis lists symptoms divided into those associated with the GI tract and those not associated with the GI tract. The odds ratio for each symptom is indicated by the circle, and associated bars represent the 95% CI. The red dashed vertical line at aOR = 1.0 indicates no association. The percentage of surveys reporting each symptom is provided to the left of these bars. aOR and the 95% CIs are listed to the right of the bars. Analyses where sample size was insufficient are listed as “Too few reports.” (B and C) Identical data to (A) where (B) lists participants with negative paired OP swabs for SARS-CoV-2 RNA, and (C) lists participants with positive paired OP swabs for SARS-CoV-2 RNA.
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